Synthesis of histamine dihydrochloride

ABSTRACT

The invention disclosed herein relates to the preparation of pharmaceutical grades of histamine dihydrochloride using a two step non-enzymatic synthetic method. The invention disclosed herein describes the synthesis of histamine dihydrochloride by the non-enzymatic decarboxylation of histidine and the step-wise conversion of the decarboxylated product to the dihydrochloride salt form. The invention disclosed herein considers a final product of histamine dihydrochloride containing less than each of the following: 0.8% L-histidine HCl monohydrate, 0.1% individual chromatographic impurities, and 2% total impurities, to be acceptable for pharmaceutical use.

BACKGROUND OF THE INVENTION

Histamine is a compound possessing significant biological activitymediated by pharmacological receptors. Histamine has long beencontemplated as a molecule having primarily negative biological effects.Recently, however, new uses for histamine as a powerful pharmaceuticalagent have come to light. For example, histamine has been used inconjunction with interferon-alpha to activate NK cells in the presenceof monocytes. See U.S. Pat. No. 5,728,378. To take full advantage of thetherapeutic properties of histamine, it is necessary to obtain largequantities of the compound in a pharmaceutical grade.

Histamine occurs widely in nature as a result of putrefactive processesand a derivative, histamine dihydrochloride, is sold commercially foruse as a standard in assays and as a component in certain allergydiagnostic kits. The source of this histamine is often a natural one andas such contains a variety of contaminants that render it unsuitable forpharmaceutical use. There are also synthetic protocols for the synthesisof histamine dihydrochloride known in the art.

Histamine dihydrochloride can be conveniently synthesized by exploitingthe decarboxylation of histidine. Using this synthesis pathway,histidine is decarboxylated and subsequently treated to form thedihydrochloride salt form of the molecule. For example, Hashimoto etal., discussed the preparation of histamine using cyclohexenone as acatalyst for the decarboxylation of histidine. (Hashimoto, M., et al.,Chemistry Letters, 893-896 (1986)). The Hashimoto, et al., paperreported the isolation of histamine dihydrochloride at a 95% yield,using 2-cyclohexen-1-one as the catalyst, from the reaction involvinghistidine and 1% v/v of 2-cyclohexen-1-one in 10 parts of refluxingcyclohexanol (26 hours). The Hashimoto method also teaches the use oftoluene and HCl gas bubbled through the resulting decarboxylatedsolution to precipitate out and harvest the final histaminedihydrochloride product.

Attempts to reproduce the Hashimoto procedure to generatepharmaceutically pure amounts of histamine failed. Additional amounts ofthe catalyst were required to make the procedure operative and asubstantial number of impurities were present in the final product.Moreover, those impurities were difficult to remove. In view of theseresults, it was found that the Hashimoto procedure is an unsuitablemethod for generating large quantities of pharmaceutically acceptablehistamine.

The use of acetophenone as a catalyst for the decarboxylation ofhistamine has also been reported. We recreated the method described inthe Japanese patent to Akimasa, et al., patent, Japanese Patent No.05,255,204 (1983), and used 0.26 equivalents of acetophenone and 10parts of diethylene glycol as the solvent for the decarboxylationreaction. Although the Akimasa et al. method was far more efficient inconverting histidine to histamine, it failed to consistently yield apharmaceutical grade product. Like the final product using the Hashimotomethod, impurities were observed in the final product made using theAkimasa method during the HPLC analysis.

Although the conditions with acetophenone and diethylene glycol lookedpromising, there existed a problem related to the work-up. Bothhistamine free base and the dihydrochloride salt are readily soluble inwater, therefore, it was difficult to utilize any extraction techniqueto separate the product from the diethylene glycol solvent, which wasusually removed by a water extraction. Furthermore, the histaminedihydrochloride was also readily soluble in diethylene glycol, thus thedirect isolation by filtration was also impossible.

The reaction conditions of Takano et al., involving pentan-3-one werealso recreated. (Heterocycles, 6:1167 (1977)). The results from theseexperiments showed no improvement over the acetophenone conditionsdescribed above.

A consistent source of pharmaceutical grade histamine is required,especially in view of the new-found pharmaceutical applications forhistamine. The standard methods used by the art wherein histamine ispurified from natural sources, fail to yield histamine of a sufficientlyhigh grade for pharmaceutical uses. Moreover, the synthetic methodspracticed in the art also fail to yield histamine of a sufficiently highgrade. Accordingly, there is a need in the art for an improved method bywhich to produce pharmaceutical grade histamine dihydrochloride.

SUMMARY OF THE INVENTION

The invention disclosed herein relates to the preparation ofpharmaceutical grades of histamine dihydrochloride using a two stepnon-enzymatic synthetic method. One embodiment of the invention is amethod for the synthesis of histamine dihydrochloride comprising:decarboxylating a L-histidine containing solution, whereby a histaminecontaining solution is formed in the absence of a decarboxylatingenzyme; forming a histamine monohydrochloride containing solution fromthe histamine containing solution; and forming a histaminedihydrochloride containing solution from the histamine monohydrochloridecontaining solution.

One aspect of this embodiment further comprises triturating thehistamine containing solution, for example, the histamine containingsolution can be triturated with a methylene chloride solution. Inanother aspect of this embodiment, the histamine monohydrochloridecontaining solution is formed by addition of an effective amount ofhydrochloric acid in an isopropanol solution. For example, the effectiveamount of hydrochloric acid is about 0.1 to 0.9 molar equivalents ofhydrochloric acid to histamine free base. In another example, theeffective amount of hydrochloric acid is about 0.6 molar equivalents ofhydrochloric acid to histamine free base. Still another aspect of thisembodiment further comprises the step of isolating a pharmaceuticalgrade of histamine dihydrochloride from the histamine dihydrochloridecontaining solution.

Another embodiment of the invention disclosed herein is a method forsynthesizing a pharmaceutical grade of histamine dihydrochloridecomprising: decarboxylating a L-histidine containing solution, whereby ahistamine containing solution is formed in the absence of adecarboxylating enzyme; forming a histamine monohydrochloride containingsolution from the histamine containing solution; forming a histaminedihydrochloride containing solution from the histamine monohydrochloridecontaining solution; and isolating the histamine dihydrochloride fromthe histamine dihydrochloride containing solution.

In one aspect of this embodiment, the histamine dihydrochloride containsequal to or less than each of the following: 0.8% L-histidine HClmonohydrate, 0.1% individual chromatographic impurities, and 2% totalimpurities.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a reaction method taught in the art.

FIG. 2 shows the method of the invention disclosed herein discussed in

Examples 5 and 6.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein relates to the preparation ofpharmaceutical grades of histamine dihydrochloride using a two stepnon-enzymatic synthetic method. The invention disclosed herein describesthe synthesis of histamine dihydrochloride by the non-enzymaticdecarboxylation of histidine and the step-wise conversion of thedecarboxylated product to the dihydrochloride salt form. The inventiondisclosed herein considers a final product of histamine dihydrochloridecontaining less than each of the following: 0.8% L-histidine HClmonohydrate, 0.1% chromatographic impurities (defined below), and 2%total impurities, to be acceptable for pharmaceutical use.

Synthetic methods of synthesizing histamine dihydrochloride known in theart fail to yield product of a sufficient purity to be used as apharmaceutical compound. FIG. 1 shows a decarboxylation method taught inthe art. The method steps of the invention disclosed herein are shown inFIG. 2.

Prior art methods were used to generate histamine dihydrochloride fromL-histidine starting material in an attempt to generate pharmaceuticalgrades of synthetic histamine dihydrochloride. The starting material wasreacted with the prior art catalyst α-tetralone and cyclohexane. Aftercompletion of the reaction, the sample was cooled and hydrochloric acidwas bubbled into the solution to convert the histamine free base intothe dihydrochloride salt form. The precipitate that formed was filtered,washed, and dried. The final product produced by the prior art methodwas found to contain an unacceptably high number of contaminants.

The crude material produced using the prior art method had a purity of92-94% with one major impurity at 3-5% and five to eight otherimpurities at ≧0.1%. Additional purification steps or recrystalizationswere performed and were substantially effective at removing most ofthese contaminants. Nevertheless, two unidentified impurities remainedat levels above 0.1%. These impurities eluted after the histaminedihydrochloride product and were referred to any chromatographicimpurities or contaminants. Thus, product made by this method wasunacceptable for pharmaceutical use.

In view of these results, a new procedure was designed to synthesizehistamine dihydrochloride of the desired purity. This new procedureinvolved the decarboxylation of L-histidine (α-amino-4(or5)-imidazolepropionic acid (C₆H₉N₃O₂) to yield histamine. Followingdecarboxylation, the solution containing the histamine free base wastriturated with methylene chloride to precipitate the product. Theproduct was then filtered and washed. The filtered product wassubsequently treated with hydrochloric acid in isopropanol toprecipitate a crude histamine monohydrochloride salt. This product wasfiltered and isolated. The crude salt can be subsequently purified byrecrystalization techniques or it can proceed to the final modificationstep of the present method. Next, the monohydrochloride salt was treatedagain with a hydrochloric acid/isopropanol solution to generate thehistamine dihydrochloride form of the molecule. The final form of theproduct was then decolorized and washed. These steps, known asrecrystalization, can be repeatedly extensively to yield histaminedihydrochloride of pharmaceutical purity. All steps were performed undera nitrogen gas atmosphere. The purity of the final product was analyzedthrough a number of analytical methods including HPLC analysis.

The invention disclosed herein contemplates the use of a number ofcatalysts or radical initiators to facilitate the decarboxylationreaction. An appropriate catalyst is one that will efficiently catalyzethe decarboxylation of histidine when that precursor compound is in aneutral solvent and heated for a number of hours to yield an acceptablypure final product. Electron-enriched ketones are preferred as they tendto reduce the number of impurities present in the final product. Forexample, a group of suitable catalysts comprises: benzoyl peroxide,2,2′-azobisisobutyronitrile (AIBN), 2-cyclohexen-1-one, acetophenone,4′-bromoacetophenone, benzophenone, p-nitroacetophenone,p-methylacetophenone, p-methoxyacetophenone,p-methylacetophenone/1-methyl-4-piperidone, andp-methylacetophenone/AcOH.

The decarboxylation reaction conditions promote the decarboxylation ofthe starting materials while minimizing the formation of unwantedcontaminants. The reaction conditions include conducting several methodsteps in the presence of an inert gas, for example, nitrogen; Thereaction conditions further include conducting the decarboxylation stepat a range of temperatures between about 145 to 170° C. Preferably, thereaction is carried out at a range of temperatures from about 150 to165° C., or at a range of temperatures from about 160 to 165° C.

A number of solvents are contemplated for use in the invention disclosedherein. The solvents in which certain steps of the reaction areconducted may effect the reaction time which is required to catalyze thedecarboxylation of histidine. Solvents useable in the inventiondisclosed herein include: cyclohexanol, n-methlpyrrolidinone (NMP),di(ethyleneglycol), di(ethyleneglycol)methyl ether,2-methyloxyethlether, 1-butanol, methoxyethanol, cyclohexanol/NMP (in a3:1 ratio), dimethylformamide, and tetramethylenesulfone.

Another parameter of the reaction disclosed herein is the method ofcreating the salt form of histamine by treating the reaction mixturewith hydrogen chloride. The impurity profile of the final product wasfound to be effected by the molar equivalency of acid added during theprecipitation of the monohydrochloride crude salt. It is possible tocontrol the extent of impurity formation by preparing a solution ofhydrogen chloride of a known concentration in isopropanol and treatingthe reaction mixture therewith.

A range of molar equivalents of hydrogen chloride (HCl) in isopropanol(ISA) may be used to practice the method of the invention disclosedherein. A range of about 0.01 to 2 molar equivalents may be used tocreate the salt form of histamine. Alternatively, a range of about 0.05to 1.4 molar equivalents may be used. In another alternative, a range ofabout 0.1 to 0.9 molar equivalents may be used. In yet anotheralternative, about 0.5 molar equivalents may be used. The ratio selectedto practice the invention disclosed herein should result in the ultimategeneration of a final product with an acceptable level of impurities sothat the final product may be used as a pharmaceutical composition.

The concentration of the acidic solution used to create the salt formwas not critical. For example, the concentration of HCl in ISA may rangefrom about 6 to 9 N. However, the number of moles of acid introduced iscrucial to isolating a pharmaceutically acceptable grade of the finalproduct. The addition of too much acid causes impurities to precipitatewith the monohydrochloride salt that are extremely difficult toeliminate during the subsequent formation of the histaminedihydrochloride salt. The relationship between the method of saltformation and the generation of contaminants was not appreciated in theart.

Various co-solvents may be used during the addition of HCl inisopropanol to effect precipitation of the monohydrochloride salt formof the molecule (salt precipitation). Co-solvents useable in theinvention disclosed herein include: methylene chloride, cyclohexanol,toluene and tert-butyl methyl ether (TBME).

The ultimate purity of the final product is of particular concern.Additional method steps to purify the final product are alsocontemplated. For example, recrystallization is a process of repeatedcrystallization in order to purify a substance. A number of solvents arecontemplated for use in this purification process. These solventsinclude: methyl chloride, 2-propanol, methanol, ethanol (ETOH),methanol/acetone, water, methanol/ethyl acetate, water/acetone,methanol/ethanol, water/methanol, methanol/hexane,water/methanol/acetone, methanol/methylene chloride, 2-propanol/ethanol,methanol/2-propanol, acetone/2-propanol, acetone/ethanol. From atoxicological viewpoint, a non-toxic solvent such as ETOH is preferred.

The presence of color in the various solutions obtained during thesynthesis pathway was observed. Activated carbon may be added to removesome of the color before or as a step of the recrystallization process.

The invention disclosed herein further contemplates the use ofderivitizing chemical reactions to assist in the purification ofhistamine dihydrochloride. Accordingly, it is contemplated that chemicalderivatives of various impurities would be made during the histaminedihydrochloride process of the invention disclosed herein to facilitatethe removal of those impurities. The creation of one such derivativeinvolves the addition of a tert-butoxycarbonyl group to a molecule ofinterest. Other modifying groups such as benzyloxycarbonyl groups (CBZ)are also contemplated.

The following examples discuss methods addressing the decarboxylation ofhistidine as well as the isolation of the histamine product. Alsodiscussed are methods of purification of the crude histaminedihydrocloride product using multiple recrystallization steps. Charcoalmediated decoloration is also discussed.

The efficiency of various method steps as well as the purity of thefinal product may be analyzed using the methods desired below. One ormore monitoring steps may be used to assay the efficiency of thedecarboxylation step. Alternatively, various assay methods well known inthe art may be used to analyze the purity of the final product. Anexample of such a monitoring step is the performance of thin layerchromatography (TLC), a procedure well known in the art, on variousreaction products. For example, reactions could be monitored using TLC(mobile phase: CH₃CN:H₂O:NH₄OH; 7.5:2.0:0.5; and ninhydrin spray). Thismonitoring step may be performed anytime after the decarboxylation step.

Particular embodiments of the invention are discussed in detail below.The following examples are for illustrative purposes only and should notbe interpreted as limitations of the claimed invention. There are avariety of alternative techniques and procedures available to those ofskill in the art which would similarly permit one to successfullyperform the intended invention.

EXAMPLES Preparation of Histamine Dihydrochloride

The following Examples discuss the synthesis of histaminedihydrochloride from the precursor compound L-histidine. Existinghistamine synthesis protocols, while capable of yielding histaminedihydrochloride, suffer from the limitation of producing an impure finalproduct. The Examples below discuss various improvements in histaminedihydrochloride synthesis and teach the preparation of apharmaceutically acceptable grade of histamine dihydrochloride.

Example 1 Preparation of 500 Grams of Crude Histamine Dihydrochloride

A method for the synthesis of a 500 gram sample of histaminedihydrochloride is described below.

A twelve liter (12-L), 4 necked, round bottom flask equipped with athermometer, mechanical stirrer, condenser and nitrogen bubbler wascharged with 7.5 L of cyclohexanol (the solvent), 750 grams ofL-histidine (the substrate) and 113 ml of acetophenone (the catalyst).The suspension was agitated in a nitrogen atmosphere that was maintainedthroughout the reaction.

The suspension was heated to reflux and maintained at that temperature(150-165° C.) for a minimum of 40 hours. A small sample was withdrawnfor an-in-process assay to determine the extent of histidinedecarboxylation. The suspension was cooled to below 80° C. and 1875 mlof toluene was charged. This mixture was further cooled to roomtemperature. The mixture was filtered through a Buchner funnel into afresh 12-L, 4 necked round-bottom flask.

The fresh flask containing the filtrate was equipped with a thermometer,mechanical stirrer, hydrogen chloride trap and vacuum trap, and preparedfor gaseous hydrogen chloride addition. With agitation, the solution wascooled to below 10° C. Maintaining the batch temperature below 20° C., aminimum of 441 grams (2.5 equivalents) of gaseous hydrogen chloride wascharged. Upon completion of the hydrogen chloride addition, theresulting thick yellowish suspension was agitated at room temperaturefor one hour.

The suspension was again filtered through a Buchner funnel. The filtercake was rinsed with a mixture of 375 ml of cyclohexanol and 375 ml oftoluene, followed by two 750 ml washes of toluene and two 750 ml rinsesof hexanes. The cake was dried on the filter with suction for a minimumof 30 minutes. The filter cake contained a substantial amount ofcyclohexanol which was removed through trituration.

The wet filter cake was charged to a 12-L 4-necked round-bottom flaskequipped with a mechanical stirrer and nitrogen bubbler. Ethanol (ETOH)in a volume of 7.5 L was also charged. The suspension was agitated atroom temperature for 4 hours. The suspension was filtered through aBuchner funnel and the filter cake rinsed with 400 ml of hexanes. Thefilter cake was dried in a vacuum oven at 60-65° C. overnight. Theproduct of this method produced 504 of crude histamine dihydrochloridegrams (a 56.6% yield) at 94.4% a/a purity determined using highperformance liquid chromatography (HPLC). The product was recrystallizedto improve the purity of the final product.

A 12-L, 4-necked, round-bottomed flask equipped with a thermometer,mechanical stirrer, condenser, addition funnel and nitrogen bubbler wascharged with the 503 grams of crude histamine dihydrochloride productsynthesized above. Additionally, 4.5 L of ETOH and 200 ml of water wereadded to the reaction flask to dissolve the filter cake. The suspensionwas agitated under a nitrogen atmosphere.

The suspension was heated to reflux. Maintaining the suspension underreflux, water was charged drop-wise to the suspension until most of thesolids were dissolved. The solution was cooled to below 75° C. Thesolution was charged with a mixture of 50 grams of NUCHAR SA (Westvaco,New York, N.Y.) and 50 grams of CELITE (J.T. Baker, Hayward, Calif.).This suspension was heated then heated to reflux and maintained at thattemperature for 0.5 hours. The suspension was cooled to 65-75° C. andthen filtered through a CELITE bed into a clean, dry 12-L, 4-necked,round-bottom flask. The filter cake was rinsed with a mixture of 450 mlof ETOH and 50 ml of water.

The filtered solution was slowly cooled to room temperature withstirings overnight. The solution was further cooled to 0-5° C. for 2hours. At 0-5° C., the suspension was filtered through a Buchner funnel.The filter cake was washed three times with 200 ml of ETOH chilled to0-5° C. The filter cake was dried in a vacuum oven at 60-65° C.

After recrystallization, the final product was 299 grams, (a 59.4%yield), at 99.1% a/a HPLC purity. The HPLC protocol is discussed inExample 7 below. Additional rounds of recrystallization were performedto increase the purity of the final product. However, two unknownimpurities, (RRt 1.3, 1.5) were still present above the 0.1% thresholdlevel after recrystallization.

Typically, the first impurity (RRt 1.3) was at 0.2-0.4% a/a and thesecond impurity (RRt 1.5) at 0.5-0.6%. A second recrystallization of thesample discussed above reduced the impurity levels to 0.1-0.2% and0.4-0.5%, respectively. The impurities appeared to grow when the sampleswere reanalyzed after a number of days, indicating stability concernsfor the final product. Instability of the product might explain why thewet filter cake discussed above showed 99.9% a/a HPLC purity but only99. 190 was obtained after the batch was dried. Subsequent treatmentswith dichloromethane or charcoal treatments were unable to remove theimpurities.

Example 2 Catalysts for the Decarboxylation of L-Histidine

In view of the results discussed above, a number of modifications to thesynthesis method were undertaken. These modifications sought to reducethe levels of the unknown impurities to an acceptable level. Onevariable examined concerned the nature of the catalyst used in thedecarboxylation reaction. A variety of other catalysts were examined,including acetophenone, to determine what role, if any, they play in theformation of the chromatographic impurities. Table 1 shows the catalystsused in this study. The catalysts were used at 0.3 equivalents.

TABLE 1 Survey of Decarboxylation Catalysts Reaction HPLC Purity (% a/a)Catalyst Time (h) Impurity #1 Impurity #2 Acetophenone (control) 21 4.54.0 4′-Bromoacetophenone 21 7.0 3.4 Benzophenone 21 14.5 2.9p-Nitroacetophenone 17 Decom- Decom- position positionp-Methylacetophenone 16 1.65 0.71 p-Methyloxyacetophenone 16 1.9 2.9p-Methylacetophenone/1- 7.5 3.0 2.4 methyl-4-piperidonep-Methylacetophenone/AcOH 7.5 9.6 3.5

The results in Table 1 indicate that p-methylacetophenone was superiorto acetophenone at diminishing the level of impurities found in thefinal product. In contrast, using p-methylacetophenone in conjunctionwith a base (1-methyl-4-piperidone) showed no improvement in the levelof impurity generation, while introducing an acid (acetic acid)considerably elevated the impurities found in the final product. Rather,p-methoxyacetophenone offered are advantage over acetophenone withrespect to contaminant generation, but did not produce a significantenhancement versus p-methylacetophenone upon isolating themonohydrochloride salt. The data suggest that catalysts with anelectron-deficient ketone exhibit an increase in the generation ofimpurities found in the final product, whereas electron-enriched ketonesshowed a decrease in impurity generation. Based on these results,acetophenone was replaced with p-methylacetophenone as the catalyst usedin the decarboxylation reaction of the invention disclosed herein.

Example 3 Methods of Producing Histamine Salt Forms

Another parameter explored, which concerned the generation of acceptablypure histamine dihydrochloride, involved the molar equivalency of acidadded during the precipitation of the crude salt. It is one of thesurprising discoveries of the invention disclosed herein that areduction in the amount of contaminants present in the final product isrelated to the amount of acid used to create the salt form of themolecule. In prior art procedures a quantity of 2.5 molar equivalents ofhydrogen chloride (HCl) gas was introduced into a solution containingthe decarboxylated histidine (histamine free base) to generate a crudedihydrochloride salt. The present Example examines the effect of addinga variety of molar equivalents of hydrochloric acid to the histaminefree base solution by introducing the acid dissolved in isopropanol(ISA).

A variety of HCl concentrations were dissolved in ISA and tested fortheir effects on the production of impurities. The synthesis protocolwas followed as described above except that 0.3 equivalents ofp-methylacetophenone with toluene as the co-solvent for the addition ofthe HCl were used. The HPLC protocol of Example 7 below was used todetermine the presence of impurities. The results of this range of acidconcentrations are listed in Table 2 below.

TABLE 2 Equivalents of HCl and Their Effect of Impurity Generation HPLCPurity (% a/a) Molar Equivalents Condensation of HCl/PA Impurity #1Impurity #2 Product 2 (control) 2.5 2.35 22.0 1.4 2.0 2.1 6.5 0.9 0.551.15 2.1 0.5 0.06 0.83 0.45

The results shown in Table 2 illustrate how the amount of acid chargedto the solution containing the histamine free base dramatically alteredthe level of the two impurities present in the product. The observeddecrease was likely attributable to the impurities possessing less of abasic character than that of the histamine free base. As a consequence,the histamine free base likely undergoes protonation first followed bythe impurities.

The use of 0.5 molar equivalents of HCl provided the most favorableresults with regards to limiting the levels of impurities found in theproduct. Under these conditions, the crude product isolated was themonohydrochloride salt as determined by titration for chloride content.Accordingly, to synthesize a dihydrochloride form of histamine of anacceptably high purity, an intermediate purification step involving theintentional generation of monohydrochloride salt was adopted. Using thismethod, however, it would be necessary to add an additional equivalentof HCl in a later synthesis step so as to produce the dihydrochlorideform of the molecule.

Additional experiments were performed to examine the effect of smallchanges in acid concentration on product purity and yield. The resultsof these experiments are shown in Table 3. These results were taken fromproducts formed from a 100 ml reaction mixture with 0.3 equivalents ofp-methylacetophenone and CH₂Cl₂ as the co-solvent. The selection ofCH₂Cl₂ is discussed in detail in Example 4.

TABLE 3 Small Variations of Acid Equivalents and Their Effect on ProductFormation CRUDE SALT FINAL PRODUCT HPLC Purity HPLC Purity (% a/a) (%a/a) HCl Yield Impurity Yield Impurity (eq.) % Impurity #1 #2 % Impurity#1 #2 0.57 43.3 0.05 0.14 57.7 0.03 0.07 0.67 50.6 0.06 0.17 61 0.050.10 0.76 53.5 0.09 0.20 59 0.05 0.1

The equivalency window was narrowed to determine the effect thatrelatively small variation in the amount of acid had on the impurityprofile in the crude salt. The data shown in Table 3 support theprevious observation that a decrease in the quantity of acid chargedresults in a decrease in the amount of impurities found in the finalproduct, as well as a decrease in the yield. In future experiments 0.6molar equivalents of HCl versus the starting material was used. Forlarger amounts of product using larger amounts of starting material, theamount of acid required is 0.85 molar equivalents of HCl per mole offree base, as determined by assay. This amount of HCl calculatedrepresents approximately 0.6 molar equivalents versus the startingmaterial of L-histidine.

Example 4 Co-solvents for Use During Salt Formation

The next variable examined to improve the synthesis of histaminedihydrochloride concerned co-solvent used during the acid addition stepof the procedure. Previously, toluene was used as the co-solvent. Toexplore the possible effect of the co-solvent on the purity of the finalproduct, a variety of co-solvents were used in the precipitation step.As above, the purity of the resulting samples was assayed using the HPLCmethod described in Example 7. The results are shown in Table 4.

TABLE 4 The Effect of Methylene Chloride and Other Co-solvents on FinalProduct Purity HPLC Purity (% a/a) Co-solvent % Yield Impurity #1Impurity #2 Toluene (control) 49.5 0.13 0.31 CH₂Cl₂ 45.9 0.10 0.15 TBME51.9 0.33 0.51 None 43.5 0.12 0.18

The reaction conditions for the results produced in Table 4 werep-methylacetophenone present in 0.3 equivalents, 0.6 equivalents ofHCl/IPA and 5 parts of co-solvent for precipitation. The results inTable 4 show that methylene chloride provides superior results withrespect to impurity formation as compared to other co-solvents.

Example 5 Preparation of Crude Histamine Monohydrochloride

The procedure described below teaches the preparation of histaminemonohydrochloride. A two liter (2-L), 3-necked, round-bottomed flask(the reactor) was equipped with a thermometer, mechanical stirrer,condenser and nitrogen purge system was charged with 1 L ofcyclohexanol, 100 gm of L-histidine and 25.9 ml of p-methylacetophenone.Cyclohexanol has a melting point of 22—22° C. and may require heating togenerate a liquid that can be transferred to the reactor. The suspensionhad a white coloration, with a temperature of between 20-25° C. and avolume of 1050 ml. The suspension was agitated in the presence of anitrogen atmosphere that was maintained throughout the reaction.

The suspension was heated to reflux (160-165° C.) and maintained underreflux for 30 hours. A small sample was withdrawn to determine whatpercentage of the starting material had been decarboxylated. Thesuspension should contain ≦1% a/a L-histidine. In the event of anincomplete reaction, continue heating the suspension at reflux for anadditional 3-5 hours and then resample. The formation of a clear,homogeous solution indicates the consumption of all the startingmaterial and the completion t the decarboxylation reaction.

Once the reaction was complete, the suspension was cooled to about20-25° C. Then the reactor was charged with 300 ml of methylenechloride. This mixture was further cooled to room temperature. Themixture was filtered through a Buchner funnel into another 2-L 3-neckedround-bottomed flask. The first reactor was then washed twice with 100ml methylene chloride that was then used to rinse the filter. Thisfiltration step removed any residual L-histidine.

The second reactor containing the filtrate was equipped with athermometer, mechanical stirrer, addition funnel and nitrogen purgesystem. After the washing step and the re-establishment of the nitrogenatmosphere in the reactor, the filtrate was heated to 30-35° C. Analiquot of the solution was withdrawn and assayed for the content ofhistamine free base. The results from the assay were used to calculatethe amount of acid required to generate the monohydrochloride salt. Theamount of acid required was 0.85 molar equivalents of HCl per mole ofhistamine free base.

With vigorous agitation, 50.5 ml of a 7.65M HCl isopropanol (HCl/ISA)solution was added dropwise at a rate where the temperature of thesolution did not exceed 40° C. Given the exothermic nature of thismethod step, addition of the HCl/ISA solution occurred over the time ofan hour. The resulting light beige suspension was allowed to cool to20-25° C. over 1 hour and agitated for a minimum of 2 hours. The 7.65 MHCl in isopropanol solution was prepared by bubbling 27.9 g of HCl gasinto 100 ml of isopropanol chilled to 5-10° C.

The cooled suspension was filtered through a Buchner funnel under astream of nitrogen and the filter cake rinsed three times with 100 ml ofa 1:1 methylene chloride/cyclohexanol solution. The filter cake was thenwashed three times with 100 ml of methylene chloride. Since themonohydrochloride salt was readily soluble in water, the humidity of thelaboratory may have an effect on the yield of the product. Therefore,exposure of the filter cake to moisture during the filtration step wasminimized by performing the operation under a stream of nitrogen.

The wet filter cake was then charged to a 1L, 3-necked round bottomflask equipped with a thermometer, mechanical stirrer and nitrogen purgesystem for methylene separation. The solid was suspended in 75 ml ofmethylene chloride and agitated for 1 hour under nitrogen. The methylenetrituration assisted in the removal of residual cyclohexanol and enabledthe product to be dried more effectively, as was seen in the subsequentsteps described below.

The suspension, under a stream of nitrogen, was filtered and the solidmaterial was washed twice with 75 ml of methylene chloride. The filtercake was dried in a vacuum oven at 55-60° C. for 16 hours.

Table 5 below shows the results of the method described in this Example.This method was practiced three times and the product yields from eachwere compared.

TABLE 5 Crude Yields of Histamine Monohydrochloride Experiment 1Experiment 2^(‡) Experiment 3^(‡) Weight of Dry Solid^(†) 50.28 52.2950.28 % Crude Yield 52.9 55.0 52.9 ^(†)Weight of solids andcorresponding percentage yields were corrected for solvent content.^(‡)In Experiments 2 and 3, the filter cake was dried for 8 rather than16 hours.

Example 6 Preparation of Histamine Dihydrochloride by Decarboxylation ofL-Histidine

Example 6 shows a procedure for the synthesis of histaminedihydrochloride from the monohydrochloride precursor product producedwith the method of Example 5.

A one liter (1 L) three-necked, round bottom flask (the reactor)equipped with a mechanical stir bar, an addition funnel, a condenser, anitrogen purge system, and thermometer was placed in a heating mantle.The reactor was charged with 40 grams of histamine monohydrochloride, 32ml H₂O (distilled), and 280 ml of a 1X ETOH solution consisting of 99.5%ETOH and 0.5% toluene. A nitrogen atmosphere was maintained throughoutthe reaction as the histamine monohydrochloride salt was veryhygroscopic.

The next step of the method entailed the addition of a HCl/ISA solutionto convert the histamine monohydrochloride salt to the dihydrochlorideform. To the reactor was added 41.5 ml of 6.85M HCl/ISA solution of 1.05equivalents). As discussed above, the addition of the acid solution wasexothermic, therefore, the acid was added over a 15 minute time frame.During the initial stages of the acid addition, a clear solution wasgenerated, however this quickly returned to a thick off-white suspensionafter approximately 75% of the acid was introduced.

After addition of the acid was complete, the resulting thick, off-whitesuspension was heated to reflux (78-80° C.) in an oil bath. The solidmatter in the suspension gradually dissolved to form an amber solution.Once the solid matter was completely dissolved, the reactor was removedfrom the oil bath. The reactor was then charged with NUCHAR SA charcoal(2 grams) and CELITE (2 grams). This suspension was heated to reflux for25 minutes. Maintenance of temperature was important as the productwould precipitate at about 60° C.

The hot, black suspension was filtered through a bed of CELITE into afresh 1L, 3-necked, round bottom flask equipped with a mechanicalstirrer and thermometer. The CELITE bed served as a barrier to preventthe flow of the charcoal through the filtering unit. The fresh reactorhad been pre-heated in an oil bath and the charging of the reactor alsooccurred in this oil bath.

The first reactor containing the reaction mixture was rinsed twice with40 ml of ETOH 1 X solution at a temperature of 60-65° C. This solutionwas filtered and added to the filtrate produced above. The addition ofthe rinse volume produced some precipitate in the filtrate. The totalvolume of solution was then agitated by stirring at 60-65° C. for 30minutes.

The suspension (histamine dihydrochloride) was then slowly cooled to 25°C. over 1 hour, and agitated at 20-25° C. for 2 hours and then cooled to0-5° C. for 2 more hours. The suspension was then filtered under astream of nitrogen and the filter cake washed three times with 40 ml ofcold ETOH 1 X. The filter cake was then weighed and dried in a vacuumoven at 55-60° C. for 16 hours. The results of three differentexperiments converting histamine monohydrochloride to thedihydrochloride salt form are shown in Table 6.

TABLE 6 Yields of Histamine Dihydrochloride Experiment 4 Experiment 5Experiment 6 Weight of Wet Cake 48.8 45.6 46.4 (grams) Weight of DrySolid 34.9 33.3 33.6 (grams) % Yield^(˜) 70 66.7 67.4 ^(˜)The percentyield figure was based on the histamine monohydrochloride corrected forsolvent content.

Example 7 An HPLC Method to Assay. Identify and Determine Purity ofHistamine dihydrochloride

This example discusses the use of HPLC to quantitate and identifyhistamine dihydrochloride and to quantitate related substances anddegradants in the final product. The method employed a complete HPLCsystem with gradient and UV detection capabilities. For chromatographicpurity determinations, a system containing a computerized dataacquisition system was utilized. Other equipment used included: a WatersSymmetry C-18,5μm, 4.6×350 mm column; an analytical balance with 0.01 mgor 0.01 g resolution; volumetric glassware; and a column heater.Reagents and standards used included: a USP histamine dihydrochloridereference standard or equivalent; methanol, HPLC grade; acetonitrile,HPLC grade; 1-heptane sulfonic acid, sodium salt, Fisher Scientific(Pittsburgh, PA) HPLC grade or equivalent; sodium phosphate, monobasic,monohydrate, ACS reagent grade; D-, L-histidine monohydrochloride,monohydrate, (Sigma, St. Louis, Mo.); 1 N sodium hydroxide solution; 1 Nhydrochloric acid solution; purified water; and benzyl alcohol, ACSreagent grade or equivalent.

Two mobile phase buffers were prepared. Mobile Phase A (MPA) contained0.02 M sodium phosphate monobasic and 0.005 M heptanesulfonic acid, pHadjusted to 3.0. Mobile phase B (MPB) contained acetonitrile(ACN)/methanol (MeOH): 20/15 (v/v). standards and samples were preparedfor the assay and chromatographic purity determinations. The assaystandards involved the preparation of histamine dihydrochloride standardsolutions at three concentrations, 0.88 mg/ml, 0.80 mg/ml, and 0.72mg/ml. DL-histidine monohydrochloride, monohydrate standards wereprepared at 0.008 mg/ml. Similarly, assay samples were prepared induplicate to contain 0.8 mg/ml of synthetically produced histaminedihydrochloride while limit of quantitation (LOQ) solution was preparedat 0.0006 mg/ml of histamine dihydrochloride. The sensitivity of themethod for Histamine has been determined to be 0.07% for the limit ofquantitation and 0.03% for the limit of detection. Photodiode array peakpurity studies have demonstrated the specificity for histamine.

Following preparation of the various standards and samples, the HPLCsystem was equilibrated. Once equilibrated, the flow rate from the wasteline was checked at the initial condition setting (i.e., 10% MPB at 1.5ml/minute). The flow rate was 1.5 ml/minute ±0.15 ml/minute. A waterblank injection was made after the system equilibrated to condition thecolumn prior to the start of the assay.

Once these preparations were complete, the resolution solution of 0.7mg/ml±0.1 mg/ml histamine dihydrochloride was injected. The resolution“R” between a 1 mg/ml benzyl alcohol solution peak and histamine peakswas calculated. Further, this process was repeated five (5) times and astandard deviation was calculated.

For the assay, a standard curve was generated. The standard check wasperformed every four to six sample injections and fell within thefollowing parameters: the tailing factor was not >2.0; the resolutionwas >1.5, the relative standard deviation of the histamine peakresponses was not >2.0%; and the correlation coefficient of the standardcurve was not less than 0.995.

To calibrate the chromatographic purity, a single injection of theresolution solution was made. The resolution “R” between benzyl alcoholand the histamine peaks was calculated and so was the tailing factor ofthe histamine peak. Since the resolution and tailing factors met thespecifications, three consecutive injections of the LOQ sample wereperformed.

The relative standard deviation for the three histamine peak responseswere calculated. In general, the tailing factor was not >2.0, theresolution was greater than 1.5, and the relative standard deviation ofthe histamine peak responses was not greater than 10%.

Since the above parameters were met, the final histamine dihydrochloridesamples were tested. The operating parameters for the HPLC are listed inTable 7. The gradient parameters are listed in Table 8.

TABLE 7 Operating Parameters Flow Rate: 1.5 ml/minute Injection Volume:20 μl Detection: 212 nm Column: Waters Symmetry C-18, 5 μm, 4.6 mm × 250mm Column Temperature: 50° C. Assay Concentration: 0.8 mg/ml histaminedihydrochloride Run Time: about 30 minutes Mobile Phase A: buffersolution Mobile Phase B: ACN/MeOH 20/15 (v/v)

TABLE 8 Gradient Parameters Time (min) % Mobile Phase B Flow Rate(ml/min) 0 10 1.5 20 30 1.5 21 10 1.5 30 10 1.5 Retention times:histidine = approximately 3 minutes histamine = approximately 12 minutes

Use of this analytical system provided the method required to determinethe purity of the histamine dihydrochloride sample produced in theaforementioned examples.

Example 8 HPLC Analysis of Histamine Dihydrochloride Product

The histamine dihydrochloride products from Example 6 were subjected tothe HPLC analysis described in Example 7 to determine the purity of thesamples and to establish whether the final products met the criteria ofpurity set for the method of the invention disclosed herein. For use asa pharmaceutical agent, the histamine dihydrochloride must possessminimal chromatographic impurities. Individual impurities found atlevels above 0.1% a/a generally require toxicological qualification.Three lots of histamine dihydrochloride were generated using the methodsof Examples 5 and 6. Their purity is described in Table 9.

TABLE 9 HPLC Analysis Results Specification/ Experiment Description ofImpurities Found Experiment 1 L-histidine HCl monohydrate <0.8% w/w; Notdetected Individual chromatographic impurities <0.1% w/w Impurity #1<0.05% w/w Impurity #2 0.13% w/w Total chromatographic impurities <2.0%w/w; 0.2% w/w Experiment 2 L-histidine HCl monohydrate <0.8% w/w; Notdetected Individual chromatographic impurities <0.1% w/w Impurity #1<0.05% w/w Impurity #2 0.10% w/w Total chromatographic impurities <2.0%w/w; 0.2% w/w Experiment 3 L-histidine HCl monohydrate <0.8% w/w; Notdetected Individual chromatographic impurities <0.1% w/w Impurity #1<0.05% w/w Impurity #2 0.06% w/w Total chromatographic impurities <2.0%w/w; 0.1% w/w

The results described in Table 9 show that the final histaminedihydrochloride product falls within acceptable standards set for theinvention disclosed herein. First, the level of Impurity #1 was found tobe below the limit of quantitation for the assay. Second, Impurity #2was found at levels slightly above the 0.1% threshold and will thereforebe qualified through toxicological testing. The specification level forimpurity has been established as <0.2% w/w. These results show that thesynthesis method of the invention disclosed herein provides a means tosynthesize a pharmaceutically acceptable form of histaminedihydrochloride.

Conclusion

The invention disclosed herein describes a novel, non-enzymatic methodfor producing pharmaceutical grade histamine dihydrochloride. Onesignificant advantage of the method described herein is that it yieldshistamine dihydrochloride at a purity level higher than is otherwisepresently available.

Finally, the forgoing examples are not intended to limit the scope ofthe present invention, which is set forth in the following claims. Inparticular, various equivalents and substitutions will be recognized bythose of ordinary skill in the art in view of the foregoing disclosure,and these are contemplated to be within the scope of the disclosedinvention.

What is claimed is:
 1. A method for the synthesis of histaminedihydrochloride comprising: forming a histamine monohydrochloridecontaining solution from a decarboxylated L-histidine-containingsolution by addition of hydrogen chloride; and forming a histaminedihydrochloride containing solution from the histamine monohydrochloridecontaining solution by addition of hydrogen chloride, wherein theresulting histamine dihydrochloride comprises less than 2% totalimpurities as measured by HPLC analysis.
 2. The method of claim 1,further comprising triturating the histamine containing solution.
 3. Themethod of claim 2, wherein the histamine containing solution istriturated with a methylene chloride solution.
 4. The method of claim 1,wherein the histamine monohydrochloride containing solution is formed byaddition of hydrochloric acid in an isopropanol solution.
 5. The methodof claim 4, wherein the hydrochloric acid is about 0.1 to 0.9 molarequivalents of hydrochloric acid to histamine free base.
 6. The methodof claim 4, wherein the hydrochloric acid is about 0.6 molar equivalentsof hydrochloric acid to histamine free base.
 7. The method of claim 1,further comprises the step of isolating a pharmaceutical grade ofhistamine dihydrochloride from the histamine dihydrochloride containingsolution.
 8. A method for synthesizing a pharmaceutical grade ofhistamine dihydrochloride comprising: forming a histaminemonohydrochloride containing solution from a decarboxylatedL-histidine-containing solution by addition of hydrogen chloride; andforming a histamine dihydrochloride containing solution from thehistamine monohydrochloride containing solution by addition of hydrogenchloride; isolating the histamine dihydrochloride from the histaminedihydrochloride containing solution, wherein the resulting histaminedihydrochloride comprises less than 2% total impurities as measured byHPLC analysis.
 9. The method of claim 8, wherein the histaminedihydrochloride contains equal to or less than each of the following:0.8% L-histidine HCl monohydrate, and 0.1% individual chromatographicimpurities.